CN101624234B - Microorganism water treatment agent in culturing water and preparation technique thereof - Google Patents
Microorganism water treatment agent in culturing water and preparation technique thereof Download PDFInfo
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- CN101624234B CN101624234B CN2009100318441A CN200910031844A CN101624234B CN 101624234 B CN101624234 B CN 101624234B CN 2009100318441 A CN2009100318441 A CN 2009100318441A CN 200910031844 A CN200910031844 A CN 200910031844A CN 101624234 B CN101624234 B CN 101624234B
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- Farming Of Fish And Shellfish (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
本发明涉及“养殖水体用微生物水质净化剂及制备工艺”，属于动物养殖技术领域。由克雷伯氏菌(Klebsiella)AN-4、枯草芽孢杆菌(Bacillus subtilis)、红螺菌(Rhodospirillum)和植物乳杆菌(Lactobacillus plantarum)组成。采用豆饼粉和糖蜜为原料，进行深层发酵，生产出含活菌数高，能有效净化水质的净水剂。该产品成本低廉、绿色环保，克服了当前水质净化剂稳定性差，抗逆性不强等问题，可用于大规模工业化生产，为水产养殖水体的净化提供一种新型微生物制剂。
The invention relates to a "microbial water quality purifier for aquaculture water body and a preparation process", and belongs to the technical field of animal aquaculture. Consists of Klebsiella AN-4, Bacillus subtilis, Rhodospirillum and Lactobacillus plantarum. Soybean cake powder and molasses are used as raw materials for submerged fermentation to produce a water purifying agent with a high number of viable bacteria that can effectively purify water. The product is low in cost, green and environmentally friendly, and overcomes the problems of poor stability and low stress resistance of current water purifiers. It can be used in large-scale industrial production and provides a new type of microbial preparation for the purification of aquaculture water.
Description
Technical field
The present invention relates to microorganism water treatment agent in culturing water and preparation technology thereof, belong to the animal cultivation technical field.
Background technology
Studies show that in current aquaculture process, because bait utilization is low, residual bait, cultivated animals ight soil discharge problems such as too much, cause the eutrophication of water body and C, N, P enrichment pollution and the bed mud redox potential of bed mud to reduce.Water body eutrophication degree increases, and can cause ammonia nitrogen and nitrite to exceed standard, and aquatic animal is produced toxic action.The discharging of a large amount of breeding wastewaters has caused tremendous influence to surrounding enviroment, and water environment worsens, and red tide takes place frequently, and the eubiosis and species diversity are also destroyed.The deteriorating water quality of culturing the waters is also given China's fishery economic massive losses that arrived.Therefore, how to solve the ammonia and nitrogen pollution problem in the aquaculture water, become the research focus of purifying aquatic water.
The microorganism recovery technique is mainly used in aquaculture in the original position reparation of breeding environment, mainly handles the organic contamination of bed mud and the eutrophication problem of water body.Studies have shown that microorganism is applied to has the degraded organic matter in the aquaculture water, the decomposition and inversion objectionable impurities is stablized water pH value, suppresses the effect of pathogenic micro-organism.At present, the probiotics commodity are more on the market, and its main probiotics has photosynthetic bacterium, budding fungus, bacillus category, actinomycetes, milk-acid bacteria, nitrobacteria, denitrifying bacterium, efficacious microbial colony etc.Their effects in aquaculture are mainly reflected in nutritive property, immunological characteristic and improve the ecological environment aspect three.In aquaculture, obtain certain effect though be applied to the probiotics of other field, but these bacterial strains are not the intrinsic bacterium in the water body environment, differ and in water body environment and cultivated animals enteron aisle, survive surely, the successful reproduction that more differs surely becomes dominant microflora to be regulated, and research thinks that the microorganism of adding is easy to along with the time wither away gradually.Simultaneously, even from aquaculture water environment or hydrocoles enteron aisle, isolate indigenous bacterium, equally can not reach expected effect in default of enough mature theory guidances as probiotics yet.In addition, single microorganism can utilize the kind of substrate limited, the organism in the water body thoroughly can not be degraded, and therefore different microorganisms need be carried out compatibility, collaborative playing a role.
Current, in aquaculture water purification of water quality field, the bacterial classification that has been applied to produce has kind more than ten, mostly is from external import or is applied to the bacterial classifications of livestock and poultry.The indigenous bacterium that derives from aquaculture water seldom.Reinforcement is fit to the probiotics product that aquaculture water uses to deriving from the fundamental research of aquaculture water microorganism for Development and Production, further improves the aquatic animal living environment and has active operation significance.Therefore, this patent is according to the degradation characteristic of bacterial classification, compound klebsiella, subtilis, redly patrols bacterium and four kinds of bacterium of plant lactobacillus.Wherein, klebsiella AN-4 is that this laboratory separation screening from the freshwater fish culturing water body obtains, and its biological nature is furtherd investigate, has grasped bacterial strain to organic degradation characteristic; Safety testing is the result show, the used klebsiella AN-4 of this product is to aquatic animal toxicological harmless and pathogenic.Other three kinds of bacterium are available from Yangzhou Bioisystech Co., Ltd of green section.The best use of condition that this product bacterial strain uses therefor purifies water meets aquaculture water surrounding conditional request, illustrates that bacterial strain is adapted at using in the aquaculture.At present, domestic report and the related products that is not applied to purification of water quality as yet relevant for klebsiella.Klebsiella is this product main bacteria seed, through taming the effect with good degradation of ammonia nitrogen, suppresses denitrification process in conjunction with subtilis and rhodospirillum, can effectively purify water, and is aided with plant lactobacillus, can strengthen aquatic health.Pilot product is 15000 kilograms of the amounts of promoting the use of, popularizing area is above 15000 mu, promoting the checking result shows, this product has very strong water quality regulation effect, can significantly reduce aquaculture water organic pollutant (as ammonia nitrogen, nitrite, hydrogen sulfide etc.), use during this product, the pond culture organic pollutants in water body remains on lower level always, and, the after stain index of stopping using can not have a rebound yet, this point obviously is better than current market and sells most of water conditioner product, therefore obtains a large amount of favorable comments.Whole high temperature season is used the breed pond of this product, and quantity of exchanged water obviously reduces, and has saved water resources, has reduced the pollution of aquaculture water to outside water surrounding, meets the Sustainable development requirement; Simultaneously, because the improvement of condition of water quality, the cultivated animals living environment takes a turn for the better, and sickness rate, mortality ratio obviously reduce, the usage quantity of medicine significantly reduces, or even zero use, has not only reduced pollution, also meet food safety requirements, guaranteed quality product, improved culture benefit conscientiously.
Summary of the invention
Technical problem
The purpose of this invention is to provide " microorganism water treatment agent in culturing water and generation preparation technology ", by filtering out the bacterium that can effectively purify water, be aided with relevant bacterial strain and form complementary action, adopting soybean cake powder and molasses is raw material, carry out submerged fermentation, produce and contain the viable count height, the water purification agent that can effectively purify water.Problems such as this low cost product, environmental protection have overcome current water quality cleansing agent poor stability, and resistance is not strong can be used for large-scale industrial production, for the purification of aquaculture system provides a kind of novel microorganism preparation.
Technical scheme:
The preparation method of microorganism water quality cleansing agent of the present invention the steps include:
(1) complex microorganism preparations is selected bacterial classification for use.Complex microorganism preparations is by klebsiella AN-4 (Klebsiella,), subtilis (Bacillus subtilis, code name CICC 10074), rhodospirillum (Rhodospirillum, code name ACCC02976) and plant lactobacillus (Lactobacillus plantarum, ACCC 10643) form.Klebsiella AN-4 is obtained by this laboratory screening, and the NCBI accession number is EU888474, and biological company limited of green section buys from Gaoyou, Yangzhou for subtilis, rhodospirillum and plant lactobacillus.
(2) klebsiella AN-4 and subtilis are inserted triangular flask (substratum: peptone 10g/mL, extractum carnis 5g/mL, NaCl 5g/mL respectively.Inoculative proportion 1%), shaking culture 24h.Rhodospirillum inserts Kolle flask (substratum: NH
4Cl 1g, MgCl
20.2g, yeast extract paste 0.1g, K
2HPO
40.5g, NaCl2g, H
2O 900mL, the 0.1g/mL NaHCO of sterilization
350mL, the 50mL alcohol of sterilizing.Inoculative proportion 1%) in, illumination cultivation 48h in biochemical incubator.Klebsiella AN-4 bacterial concentration is 10
8Cfu/mL, subtilis bacterial concentration are 10
8Cfu/mL, rhodospirillum bacterial concentration are 10
9Cfu/mL.
(3) with 40%, 30%, 30% mixing by volume of above-mentioned cultured klebsiella AN-4, subtilis and rhodospirillum bacterium liquid, make stoste.Again with stoste by volume 1% ratio insert seeding tank activated seed liquid, klebsiella AN-4, subtilis and rhodospirillum fermentation with the prescription g/L of seed tank culture base are: soybean cake powder 12, brown sugar 8, Zulkovsky starch 2, yeast extract paste 0.5, K
2HPO
41, KH
2PO
42, MgSO
40.5, MnSO
40.01, CaCO
30.03 NaCl 0.5, and adjusting pH is 7.0; The inoculation back is 37 ℃ in temperature, and pressure 0.05Mpa under air flow volume ratio 1: 1 and the 200-300r/min agitation condition, cultivates 8-10h; Reach following index: thalli growth is neat; Be in logarithmic phase; 200,000,000/mL of bacteria containing amount; No living contaminants, OD
562Value 0.4, can culture transferring to the fermentor tank fermentation culture;
(4) bacterium liquid zymotechnique
Fermentation tank culture medium g/L: soybean cake powder 10, molasses 12, Zulkovsky starch 2, yeast extract paste 0.5, K
2HPO
41, KH
2 PO
42, MgSO
40.5, MnSO
40.01, CaCO
30.03 NaCl 0.5, about pH7.0-7.5, sterilization back cumulative volume must not surpass 70% of tank volume, and 30min sterilizes under 0.11Mpa pressure;
Adopt volume ratio 5-10% inoculum size to insert seed liquor, fermentation culture conditions: 37 ℃ of temperature, stirring velocity 180-200r/min, tank pressure 0.05Mpa, the interior air flow of 4h is 1 behind the culture transferring: 0.7-0.8, increase to 1: 1 gradually later on, and the general fermentation culture cycle is 18-24h, can put jar, zymocyte liquid living contaminants≤0.3% of acquisition; Bacteria containing amount 〉=3,000,000,000/ml; OD
562〉=0.8;
(5) the water quality cleansing agent microorganism is compound.
Plant lactobacillus fermentation substratum (g/L): wheat root 4, dipotassium hydrogen phosphate 0.5, ammonium sulfate 0.5, soybean cake powder 0.5, W-Gum hydrolysis sugar 5, lime carbonate 5,6.5,31 ℃ of anaerobically fermenting 72h of pH.Bacteria containing amount 〉=2,000,000,000/ml.
(6) above-mentioned complex microorganism for preparing and plant lactobacillus fermented liquid were mixed in 17: 3 by volume, add the protective material (skimmed milk+mass volume ratio 5% trehalose) of volume ratio 0.1%, packing gets final product.
Beneficial effect
This product has good degradation effect to the aquaculture water ammonia nitrogen.Experimental result is seen Fig. 1, Fig. 2, and contrast and experimental group data are two pond numerical value and get mean number.The ammonia-nitrogen content in contrast pond is risen to about 0.09mg/L by the 0.04mg/L of beginning, and rate of increase surpasses 100%, and the experiment pool ammonia nitrogen is dropped to about 0.03mg/L by 0.05mg/L, and degradation effect is remarkable.
Though the nitrite degradation degree does not have ammonia nitrogen remarkable, this product can be kept nitrite content at lower level, does not increase.Contrast pond nitrite content is risen to about 0.15mg/L by 0.1mg/L, and experiment pool is dropped to below the 0.1mg/L by 0.12mg/L.
This pilot product is 15000 kilograms of the amounts of promoting the use of, popularizing area is above 15000 mu, promoting the checking result shows, this product has very strong water quality regulation effect, can significantly reduce aquaculture water organic pollutant (as ammonia nitrogen, nitrite, hydrogen sulfide etc.), use during this product, the pond culture organic pollutants in water body remains on lower level always, and, the after stain index of stopping using can not have a rebound yet, this point obviously is better than current market and sells most of water conditioner product, therefore obtains a large amount of favorable comments.
Whole high temperature season is used the breed pond of this product, and quantity of exchanged water obviously reduces, and has saved water resources, has reduced the pollution of aquaculture water to outside water surrounding, meets the Sustainable development requirement; Simultaneously, because the improvement of condition of water quality, the cultivated animals living environment takes a turn for the better, and sickness rate, mortality ratio obviously reduce, the usage quantity of medicine significantly reduces, or even zero use, has not only reduced pollution, also meet food safety requirements, guaranteed quality product, improved culture benefit conscientiously.
Description of drawings
Fig. 1, Fig. 2 are that this patent product is to shrimp-cultivation pool water body ammonia nitrogen and nitrite nitrogen decontamination effect improving.
Fig. 3 is the microorganism water purification agent producing process
Embodiment
(1) complex microorganism preparations is selected bacterial classification for use.Complex microorganism preparations is by klebsiella (Klebsiella) AN-4, subtilis (Bacillus subtilis, code name CICC 10074), rhodospirillum (Rhodospirillum, code name ACCC02976) and plant lactobacillus (Lactobacillus plantarum, ACCC 10643) form.Klebsiella AN-4 is obtained by this laboratory screening and externally provides, and the NCBI accession number is EU888474, and biological company limited of green section buys from Gaoyou, Yangzhou for subtilis, rhodospirillum and plant lactobacillus.
(2) klebsiella AN-4 and subtilis are inserted triangular flask (substratum: peptone 10g/mL, extractum carnis 5g/mL, NaCl 5g/mL respectively.Inoculative proportion 1%), shaking culture 24 h.Rhodospirillum inserts Kolle flask (substratum: NH
4Cl 1g, MgCl
20.2g, yeast extract paste 0.1g, K
2HPO
40.5g, NaCl 2g, H
2O 900mL, the 0.1g/mL NaHCO of sterilization
350mL, the 50mL alcohol of sterilizing.Inoculative proportion 1%) in, illumination cultivation 48h in biochemical incubator.Klebsiella AN-4 bacterial concentration is 10
8Cfu/mL, subtilis bacterial concentration are 10
8Cfu/mL, rhodospirillum bacterial concentration are 10
9Cfu/mL.
(3) with 40%, 30%, 30% mixing by volume of above-mentioned cultured klebsiella AN-4, subtilis and rhodospirillum bacterium liquid, make stoste.Again with stoste by volume 1% ratio insert seeding tank activated seed liquid, klebsiella AN-4, subtilis and rhodospirillum fermentation with the prescription g/L of seed tank culture base are: soybean cake powder 12, brown sugar 8, Zulkovsky starch 2, yeast extract paste 0.5, K
2HPO
41, KH
2PO
42, MgSO
40.5, MnSO
40.01, CaCO
30.03 NaCl 0.5, and adjusting pH is 7.0; The inoculation back is 37 ℃ in temperature, and pressure 0.05Mpa under air flow volume ratio 1: 1 and the 200-300r/min agitation condition, cultivates 8-10h; Reach following index: thalli growth is neat; Be in logarithmic phase; 200,000,000/mL of bacteria containing amount; No living contaminants, OD
562Value 0.4, can culture transferring to the fermentor tank fermentation culture;
(4) bacterium liquid zymotechnique
Fermentation tank culture medium g/L: soybean cake powder 10, molasses 12, Zulkovsky starch 2, yeast extract paste 0.5, K
2HPO
41, KH
2 PO
42, MgSO
40.5, MnSO
40.01, CaCO
30.03 NaCl 0.5, about pH7.0-7.5, sterilization back cumulative volume must not surpass 70% of tank volume, and 30min sterilizes under 0.11Mpa pressure;
Adopt volume ratio 5-10% inoculum size to insert seed liquor, fermentation culture conditions: 37 ℃ of temperature, stirring velocity 180-200r/min, tank pressure 0.05Mpa, the interior air flow of 4h is 1 behind the culture transferring: 0.7-0.8, increase to 1: 1 gradually later on, and the general fermentation culture cycle is 18-24h, can put jar, zymocyte liquid living contaminants≤0.3% of acquisition; Bacteria containing amount 〉=3,000,000,000/ml; OD
562〉=0.8;
(5) the water quality cleansing agent microorganism is compound.
Plant lactobacillus fermentation substratum (g/L): wheat root 4, dipotassium hydrogen phosphate 0.5, ammonium sulfate 0.5, soybean cake powder 0.5, W-Gum hydrolysis sugar 5, lime carbonate 5,6.5,31 ℃ of anaerobically fermenting 72h of pH.Bacteria containing amount 〉=2,000,000,000/ml.
(6) above-mentioned complex microorganism for preparing and plant lactobacillus fermented liquid were mixed in 17: 3 by volume, add the protective material (skimmed milk+5% trehalose) of volume ratio 0.1%, packing gets final product.
The pond is cultured for the Gaoyou Penaeus vannamei in proof test place, outdoor breeding pond, chooses 4 cultivating pools, two control groups, two experimental group.Experimental group drop-test microecological water matter conditioning agent, experimental session water sampling every day once, detect water-quality guideline such as ammonia nitrogen, nitrite, dissolved oxygen pH, transparency, temperature, and observed and recorded each morbidity of pond culture animal and death condition, each pond economic benefit compared to the end of the year.Test arrangement carries out at high temperature season (7, August), and concrete grouping sees Table 1.
Table 1 microecological water matter conditioning agent production checking grouping
| The place | Culture object | The test tank number | The pond area | Experiment is handled |
| Gaoyou | Penaeus vannamei | 4 | 20-30 mu | Two test tanks in each two contrast ponds |
Experimental result is seen Fig. 1, Fig. 2, and contrast and experimental group data are two pond numerical value and get mean number.The ammonia-nitrogen content in contrast pond is risen to about 0.09mg/L by the 0.04mg/L of beginning, and rate of increase surpasses 100%, and the experiment pool ammonia nitrogen is dropped to about 0.03mg/L by 0.05mg/L, and degradation effect is remarkable.Illustrate that this product has good degradation effect to the aquaculture water ammonia nitrogen.Contrast pond nitrite content is risen to about 0.15mg/L by 0.1mg/L, and experiment pool is dropped to below the 0.1mg/L by 0.12mg/L.Though the nitrite degradation degree does not have ammonia nitrogen remarkable, this product can be kept nitrite content at lower level, does not increase.
This product is 15000 kilograms of the amounts of promoting the use of, popularizing area is above 15000 mu, promoting the checking result shows, this product has very strong water quality regulation effect, can significantly reduce aquaculture water organic pollutant (as ammonia nitrogen, nitrite, hydrogen sulfide etc.), use during this product, the pond culture organic pollutants in water body remains on lower level always, and, the after stain index of stopping using can not have a rebound yet, this point obviously is better than current market and sells most of water conditioner product, therefore obtains a large amount of favorable comments.Whole high temperature season is used the breed pond of this product, and quantity of exchanged water obviously reduces, and has saved water resources, has reduced the pollution of aquaculture water to outside water surrounding, meets the Sustainable development requirement; Simultaneously, because the improvement of condition of water quality, the cultivated animals living environment takes a turn for the better, and sickness rate, mortality ratio obviously reduce, the usage quantity of medicine significantly reduces, or even zero use, has not only reduced pollution, also meet food safety requirements, guaranteed quality product, improved culture benefit conscientiously.
Claims (2)
1. an aquaculture water microorganism water quality cleansing agent is made up of klebsiella (Klebsiella) AN-4, subtilis (Bacillus subtilis), rhodospirillum (Rhodospirillum) and plant lactobacillus (Lactobacillus plantarum).
2. the described aquaculture water of claim 1 application of microorganism water quality cleansing agent.
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| CN101935105B (en) * | 2010-07-22 | 2012-12-26 | 浙江工商大学 | Microbial purifying agent for aquaculture water and preparation method thereof |
| CN101940177B (en) * | 2010-08-31 | 2013-04-10 | 华南理工大学 | Application of mixed bacterial solution of photosynthetic bacteria and bacillus subtilis in cultivating turbots |
| CN102616941B (en) * | 2012-03-26 | 2013-05-15 | 大连理工大学 | Sea cucumber micro-ecological water quality regulator and preparation method thereof |
| CN104276668A (en) * | 2013-11-17 | 2015-01-14 | 上海葵亚环保科技有限公司 | Microbiological water purifying agent for ricefield eel breeding water and preparation method of microbiological water purifying agent |
| CN104986869A (en) * | 2015-07-03 | 2015-10-21 | 山东通江三达环保科技有限公司 | Natural composite plant treatment agent for domestic wastewater and preparation method thereof |
| CN106754514B (en) * | 2016-12-23 | 2019-08-13 | 钟华 | A kind of sustainable dredging removes the microbial bacterial agent preparation method of black and odorous water |
| CN108423838B (en) * | 2018-02-08 | 2020-09-11 | 普乐格（厦门）生物科技有限公司 | Microbial preparation for ecological safety water system and preparation method thereof |
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